The capsular saccharides of bacteria have been used for many years in vaccines against capsulated bacteria. As saccharides are T-independent antigens, however, they are poorly immunogenic. Conjugation to a carrier can convert T-independent antigens into T-dependent antigens, thereby enhancing memory responses and allowing protective immunity to develop. The most effective saccharide vaccines are therefore based on glycoconjugates, and the prototype conjugate vaccine was against Haemophilus influenzae type b (‘Hib’) [e.g. see chapter 14 of ref. 86].
Based on the organism's capsular polysaccharide, twelve serogroups of N. meningitidis have been identified (A, B, C, H, I, K, L, 29E, W135, X, Y and Z). Group A is the pathogen most often implicated in epidemic disease in sub-Saharan Africa. Serogroups B and C are responsible for the vast majority of cases in USA and in most developed countries. Serogroups W135 and Y are responsible for the remaining cases in USA and developed countries. A tetravalent vaccine of capsular polysaccharides from serogroups A, C, Y and W135 has been known for many years [1,2]. Although effective in adolescents and adults, it induces a poor immune response and short duration of protection and cannot be used in infants [e.g. ref. 3] because polysaccharides are T cell-independent antigens that induce a weak immune response which cannot be boosted. The polysaccharides in this vaccine are not conjugated [4]. Conjugate vaccines against serogroup C have been approved for human use, and include Menjugate™ [5], Meningitec™ and NeisVac-C™. Mixtures of conjugates from serogroups A+C are known [6-8] and mixtures of conjugates from serogroups A+C+W135+Y have been reported [9-13].
The structure of the group X capsular polysaccharide has been known since the 1970s [14] and this serogroup has been associated with a number of outbreaks of meningococcal disease, e.g. in sub-Saharan Africa and China [15,16]. Serogroup X is known to have a significantly higher attack rate than serogroup A among children below 5 years of age. Although the need for a vaccine against this serogroup has been recognised for many years [17], no effective vaccine has been developed. Conjugate vaccines against serogroup X have been proposed [17,18], but it remains unknown whether such conjugates would be immunogenic or protective against this serogroup.
Accordingly, there remains a need for conjugates of serogroup X capsular polysaccharides. Moreover, there remains a need for conjugates that can be used for vaccination against diseases caused by this serogroup.
The structure of the group X capsular polysaccharide consists of N-acetylglucosamine-4-phosphate residues held together by α1-4 phosphodiester bonds without O-acetyl groups [19]: {→4)-D-GlcpNAc-α-(1→OPO3→} (FIG. 1). Based on the similarity between their structures, a biosynthetic relationship between MenA and MenX capsular polysaccharides has been postulated [14]. MenA capsular polysaccharide tends to hydrolyse significantly in aqueous solution [20]. This instability is thought to be caused by the presence of a phosphodiester linkage involving the anomeric position and of the N-Acetyl group in position 2 of mannosamine, which can assist departure of a phosphomonoester group [21]. Another possibility is that the hydroxyl groups at position 4 of the N-acetylmannosamine subunits interact with the phosphodiester groups facilitating hydrolysis via an internal participation mechanism, as seen in the capsular polysaccharide of type 6A pneumococcus [22] and Haemophilus influenzae type b [23]. The similarity in the structures of the MenX and MenA capsular polysaccharides, particularly their common anomeric phosphodiester linkage, means that the MenX polysaccharide may suffer from similar stability problems when in aqueous solution. The intrinsic instability of the MenA capsular polysaccharide in aqueous solution means that it is often presented in a lyophilized form when contained in vaccines (e.g. in the polysaccharide vaccine Mencevax™ and the conjugate vaccines MenAfriVac™, Menveo™ and Nimenrix™). Although the MenX capsular polysaccharide could similarly be presented in a lyophilised form to improve its stability, an aqueous formulation would be more convenient. The only vaccine containing a MenA capsular polysaccharide conjugate in an aqueous formulation is Menactra™, but this vaccine requires storage at low temperatures. Such cold storage is expensive and presents practical difficulties in many of the countries where MenA and MenX outbreaks are common (e.g. sub-Saharan Africa).
Accordingly, there is a need for aqueous formulations of serogroup X capsular polysaccharides and conjugates thereof, particularly aqueous formulations that do not require refrigeration.
The development of a vaccine against MenX requires a method for polysaccharide quantification that can be used as an in-process assay and/or for the characterization of the final vaccine. The presence of phosphate groups in the MenX capsular polysaccharide means that the polysaccharide can be quantified by a colorimetric method that measures total phosphorus content [24]. However, this method lacks selectivity and therefore would not be suitable for certain in-process applications, e.g. for the analysis of polysaccharide in phosphate buffers or in the presence of phosphate-containing impurities. A more selective method would be NMR, which has been proposed already for MenX polysaccharide quantification [25]. However, this approach requires pure samples and a large amount of material. Reference 26 demonstrates an alternative approach, where the MenX polysaccharide is quantified by HPAEC-PAD, which is more sensitive than NMR and more selective than measuring phosphate content. The authors of ref 26 quantified the MenX polysaccharide by hydrolysing the sample to make glucosamine, and comparing the amount of glucosamine released against a calibration curve derived from an N-acetyl-glucosamine-6-phosphate quantitative standard. However, glucosamine may be present because of contamination, leading to inaccurate results. Accordingly, there is a need for alternative or improved methods for assaying the MenX polysaccharide, and in particular for methods that are more selective for MenX.